【病毒外文文獻】2006 Severe Acute Respiratory Syndrome Coronavirus 3a Protein Is Released in Membranous Structures from 3a Protein-Expre

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JOURNAL OF VIROLOGY Jan 2006 p 210 217 Vol 80 No 1 0022 538X 06 08 00H110010 doi 10 1128 JVI 80 1 210 217 2006 Copyright 2006 American Society for Microbiology All Rights Reserved Severe Acute Respiratory Syndrome Coronavirus 3a Protein Is Released in Membranous Structures from 3a Protein Expressing Cells and Infected Cells Cheng Huang 1 Krishna Narayanan 1 Naoto Ito 1 2 C J Peters 1 3 and Shinji Makino 1 Department of Microbiology and Immunology 1 and Department of Pathology 3 The University of Texas Medical Branch at Galveston Galveston Texas 77555 1019 and Laboratory of Zoonotic Diseases Division of Veterinary Medicine Faculty of Applied Biological Science Gifu University 1 1 Yanagido Gifu 501 1193 Japan 2 Received 11 July 2005 Accepted 3 October 2005 Severe acute respiratory syndrome coronavirus SCoV accessory protein 3a is a virus structural protein We demonstrate here that 3a protein was released efficiently in membranous structures from various cell lines expressing 3a protein A subpopulation of the released 3a protein is associated with detergent resistant membranes The presence of the YxxH9021and diacidic motifs located within the cytoplasmic tail of the 3a protein was not required for its efficient release Analysis of supernatant from SCoV infected cells with sucrose gradient sedimentation and virus capture assay indicated that the 3a protein was released from infected cells in two distinct populations as a component of SCoV particles and in membrane structures with a lower buoyant density These data provide new insights into the biological properties of SCoV 3a protein Severe acute respiratory syndrome SARS coronavirus SCoV an enveloped single stranded and positive sense RNA virus is the etiological agent of a new human disease SARS 10 18 19 22 36 During SCoV replication viral genomic sized RNA plus eight subgenomic mRNAs are pro duced 26 40 49 These subgenomic mRNAs encode the spike glycoprotein S M glycoprotein M nucleocapsid pro tein N and E protein E all of which are the viral structural proteins found in all coronaviruses 20 In addition some subgenomic mRNAs are predicted to encode several SCoV specific accessory proteins 49 Although other coronaviruses also encode accessory proteins there are no significant simi larities in the amino acid sequences among these coronavirus accessory proteins and any known cellular or viral proteins Two SCoV accessory proteins 3a and 7a proteins have been shown to be expressed in SCoV infected cells 12 17 30 47 55 57 Past studies have suggested that coronavirus accessory proteins are generally not essential for virus replication in cell culture and yet they may play roles in viral pathogenesis in vivo 9 34 35 52 53 It has been reported that apoptosis is induced in the cells expressing SCoV 7a protein 45 and those expressing SCoV 3a protein 21 The SCoV 3a protein consisting of 274 amino acids is one of the viral structural proteins that is expressed abundantly in infected cells 17 43 SCoV 3a protein has been detected in a lung specimen from a SCoV infected patient 55 and anti bodies to 3a protein are detected in convalescent SARS pa tients 46 In infected cells 3a protein is localized in intracel lular and plasma membranes and is also found in association with intracellular SCoV particles 17 55 56 SCoV 3a protein appears to be an integral membrane protein and contains two intracellular protein sorting and trafficking signals the YxxH9021 and diacidic motifs in the C terminus SCoV 3a protein dele tion mutant lacking a region encompassing both motifs fails to transport to the cell surface 47 It has been proposed that 3a protein might be incorporated into virus particles by direct association with S protein in infected cells 57 Indeed 3a protein interacts with S and M proteins in the Golgi apparatus 47 55 where virus assembly and budding occurs 14 39 50 However in the absence of S protein 3a protein is still capable of being incorporated into virus like particles VLPs in cells expressing SCoV M E and 3a proteins 43 Thus like the S protein of coronaviruses 3a protein is dispensable for VLP formation 15 16 27 whereas it can be incorporated into virus particles through its interaction with other SCoV enve lope proteins e g S and or M proteins Biological functions of 3a protein in SCoV particles and infected cells are unclear In the present study we demonstrated that the expression of 3a protein alone in cultured cells resulted in the release of 3a protein in membranous structures Furthermore analysis of culture supernatant from SCoV infected cells indicated that the 3a protein containing membranous structures were also released from infected cells MATERIALS AND METHODS Cells and virus Human embryonic kidney 293T cells and Vero E6 cells were maintained in Dulbecco modified Eagle medium supplemented with 10 fetal bovine serum L glutamine 2 mM nonessential amino acids 0 1 mM and kanamycin 100 H9262g ml Human colon carcinoma epithelium CaCo 2 cells were maintained in Minimum essential medium Eagle with 20 fetal bovine serum L glutamine 2 mM nonessential amino acids 0 1 mM and kanamycin 100 H9262g ml Cells were incubated at 37 C in 5 CO 2 The Urbani strain of SCoV was grown as described previously 17 28 Construction of plasmids for transient expression Total intracellular RNA was extracted from SCoV infected Vero E6 cells with TRIzol reagent Invitro gen according to the manufacturer s protocol SCoV 3a and 7a genes were amplified from total RNA prepared from SCoV infected cells by reverse tran Corresponding author Mailing address Department of Microbi ology and Immunology The University of Texas Medical Branch at Galveston Galveston TX 77555 1019 Phone 409 772 2323 Fax 409 772 5065 E mail shmakino utmb edu C H and K N contributed equally to this study 210 on March 9 2015 by University of Pittsburgh HSLS http jvi asm org Downloaded from scription PCR The PCR products were cloned into a mammalian expression vector pCAGGS kindly provided by Yoshihiro Kawaoka University of Tokyo Tokyo Japan resulting in pCAGGS 3a and pCAGGS 7a respectively A mu tant 3a gene containing deletion mutations in the YxxH9021 and diacidic motif 160 to 173 amino acids aa was constructed by using two step recombinant PCR The PCR product was inserted into pCAGGS yielding pCAGGS 3aH9004 The pCAGGS 3aYA pCAGGS 3aAA and pCAGGS 3aA3 mutants were con structed by using a QuikChange II site directed mutagenesis kit Stratagene The sequences of the constructed plasmids were confirmed by sequence analyses Transient protein expression 293T cells grown on 10 cm plates were trans fected with pCAGGS 3a or other plasmids as indicated in each experiment by using TransIT 293 reagent Mirus Madison WI according to the manufactur er s instructions At different times after transfection as indicated culture media were removed and cells were washed once with phosphate buffered saline Total cell lysates were prepared by using 1H11003 sodium dodecyl sulfate polyacrylamide gel electrophoresis SDS PAGE loading buffer 62 5 mM Tris HCl pH 6 8 2 SDS 10 glycerol 50 mM dithiothreitol and the samples were boiled for 5 min Preparation of antibodies and Western blotting Methods for preparation of rabbit anti 3a polyclonal antibody were reported previously 17 Anti SCoV 7a protein antibody was developed by immunizing rabbits with synthetic peptide N PSGTYEGNSPFH corresponding to aa 35 to 46 of SARS 7a protein and was affinity purified by the synthetic peptide Rabbit anti SCoV S polyclonal antibody IGM 541 was obtained from IMGENEX San Diego CA Rabbit anti SCoV M antibody AP6008b was purchased from Abgent San Diego CA Mouse anti SCoV N antiserum was provided by Xiao Hua Li at University of Texas South western Medical Center at Dallas Mouse monoclonal anti SCoV S protein antibody NR 617 was obtained through the NIH Biodefense and Emerging Infections Research Resources Repository National Institute of Allergy and Infectious Disease Goat anti actin antibody horseradish peroxidase HRP conjugated goat anti rabbit immunoglobulin G IgG HRP conjugated goat anti mouse IgG and HRP conjugated donkey anti goat IgG were purchased from Santa Cruz Biotechnology Santa Cruz CA Western blotting was per formed as described previously 17 Briefly protein samples were separated by SDS PAGE and transferred onto polyvinylidene difluoride membranes Bio Rad with the Trans Blot SD semidry transfer apparatus Bio Rad The mem branes were blotted with primary antibodies overnight at 4 C and subjected to incubation with secondary antibodies for1hatroom temperature The protein bands were visualized with ECL reagent Amersham Biosciences according to the manufacturer s instructions Purification of 3a containing membrane structures and SCoV At 48 h after transfection culture supernatants were harvested and centrifuged at 1 500 H11003 g for 10 min at 4 C to remove cell debris Samples were further clarified by filtration through 0 45 H9262m pore size syringe filters 3a containing membrane structures were then pelleted by centrifugation through a 20 sucrose cushion at 26 000 rpm for3hat4 Cbyusing a Beckman SW28 rotor To determine the buoyant density of the 3a containing membranous structures released from ex pressing cells the vesicles that were pelleted through a 20 sucrose cushion were suspended in NTE buffer 100 mM NaCl 10 mM Tris HCl pH 7 5 1 mM EDTA applied onto a 20 to 60 continuous sucrose gradient and subjected to centrifugation at 26 000 rpm for 18 h on an SW28 rotor Twelve fractions were collected from the bottom of the gradient After the densities of each fraction were measured they were then diluted with NTE buffer and centrifuged through 20 sucrose as described above The pellets were dissolved in 1H11003 SDS PAGE loading buffer and used for Western blot analysis For purification of 3a con taining membranous structures from SCoV infected cells supernatants from SCoV infected Caco2 cells were inactivated by irradiation with 2 H11003 10 6 rads by a Gammacell 60 Co source model 109A JL Shepherd and Associates San Fer nando CA as described previously 17 The inactivation of virus infectivity was confirmed by tissue culture assay After centrifugation at 1500 H11003 g for 10 min at 4 C samples were further clarified by filtration through 0 45 H9262m pore size sy ringe filters The clarified samples were partially purified by centrifugation through 20 sucrose cushions resuspended in NTE buffer and then applied onto a 20 to 60 continuous sucrose gradient and subjected to centrifugation at 26 000 rpm for 18 h by using a Beckman SW28 rotor Twelve fractions were collected and diluted in NTE SCoV virions and 3a containing membrane struc tures were pelleted through a 20 sucrose cushion as described above Membrane flotation assay Purified 3a containing pellets were suspended in NTE buffer In some experiments 3a containing pellets were suspended in NTE buffer containing 1 Triton X 100 and incubated at room temperature or 4 C for 30 min as indicated Sucrose was then added to the suspension to adjust the final concentration to 60 wt wt The samples were transferred to the bottom of Beckman SW41 centrifuge tubes and overlaid with 50 sucrose and 10 sucrose The samples were then centrifuged to equilibrium at 38 000 rpm for 18 h by using a Beckman SW41 rotor Fractions were collected from the top to the bottom of the gradient Equal amounts of each fraction were diluted in NTE buffer and analyzed by Western blotting SCoV virus capture assay SCoV virus capture assay was performed by using the methods described by others with modifications 31 Supernatants from SCoV infected Caco2 cells were centrifuged at 1 500 H11003 g for 10 min at 4 C to remove cell debris The supernatants were then applied to top of the 20 sucrose cushion and the samples were centrifuged at 26 000 rpm for3hat4 C by using a Beckman SW28 rotor The pellet was suspended in NTE buffer containing 0 3 of bovine serum albumin proteinase inhibitor cocktail Sigma and protein A Sepharose 4 Fast Flow beads Amersham Biosciences and then incubated at 4 C for 1 h After centrifugation at 750 H11003 g for 5 min the super natant was collected and then mixed with 5 H9262g of mouse anti SCoV S protein monoclonal antibody NR 167 or mouse anti H2K K D K H2K monoclonal an tibody 29 After incubation at 4 C overnight 5 H9262g of goat anti mouse IgG FcH9253 fragment specific Jackson Immunoresearch Laboratories Inc West Grove PA was added to the samples After incubation at 4 C for 3 h protein A Sepharose Fast Flow beads were added to the samples followed by further incubation for another3hat4 C After centrifugation at 750 H11003g for 5 min the supernatant was collected The pellets were suspended in NTE buffer and then washed with NTE buffer five times Finally the collected supernatant and the pellets were sus pended in SDS PAGE loading buffer and used for SDS PAGE and subsequent Western blot analysis RESULTS SCoV 3a protein is released from 3a expressing cells To examine whether expression of 3a alone results in release of 3a protein into culture supernatant 293T cells were transfected with pCAGGS 3a in which the entire 3a gene was cloned downstream of a chicken H9252 actin promoter of pCAGGS As a control the parental pCAGGS plasmid was used for transfec tion Western blot analysis using anti 3a antibody showed expression of 3a protein in pCAGGS 3a transfected cells whereas no 3a protein was detected in the control sample Fig 1A Several 3a related signals with molecular masses from 15 to 37 kDa were detected in all experiments 3a protein of 37 kDa designated 3a 1 in the present study appeared as a major 3a protein signal 3a protein of 31 kDa designed 3a 2 was consistently detected in the 3a protein transient expression experiments whereas the abundance of this protein varied among experiments data not shown To examine whether the expressed 3a protein could be released into culture medium clarified culture supernatants from 3a expressing and control cells were applied to the top of a 20 sucrose cushion and the samples were centrifuged at 26 000 rpm for3hat4 Cbyusing a Beckman SW28 rotor Western blot analysis of the pellet with anti 3a antibody clearly demonstrated that 3a 1 was re leased into the culture media of 3a expressing 293T cells Fig 1A Densitometry analysis showed that the amount of re leased 3a 1 in culture medium was ca 8 of the corresponding intracellular 3a 1 protein signal As shown in some experi ments see Fig 2A and 3 3a 2 was also readily detected in culture medium Analysis of the supernatants from 3a express ing Vero E6 cells and Caco2 cells also revealed the release of 3a protein into culture media data not shown which demon strated that the release of 3a protein from the expressing cells was not dependent on cell type In contrast to 3a protein another SCoV accessory protein 7a protein was not released into culture medium from 7a expressing 293T cells Fig 1B Also actin an abundant host protein was not found in the supernatants from both 3a and 7a expressing 293T cells strongly suggesting that the presence of 3a protein in the cul VOL 80 2006 RELEASE OF SCoV 3a PROTEIN 211 on March 9 2015 by University of Pittsburgh HSLS http jvi asm org Downloaded from ture media of 3a expressing cells was not simply due to the leaking of intracellular proteins or contamination of cell debris in the prepared samples Characterization of membranous structures carrying 3a protein Both topological prediction and biochemical studies have suggested that 3a is an integral membrane protein with three membrane spanning regions 47 55 56 A membrane flotation assay showed the migration of the released 3a protein from the bottom of the gradient containing 60 sucrose to the interface of 10 to 50 sucrose Fig 2A demonstrating that 3a protein was released in association with membranous structures When the pellet containing 3a containing mem brane structures was treated with 1 Triton X 100 at 4 C prior to flotation assay a significant portion of 3a protein was present in the interface of 10 to 50 sucrose fractions 4 5 and 6 Fig 2B and only a low level of 3a protein was detected in the bottom of the gradient fractions 10 and 11 and pellet Fig 2B A similar result was observed when the pellet carrying 3a protein was treated with 1 NP 40 at 4 C data not shown When the pellet containing 3a containing membrane struc tures was treated with 1 Triton X 100 at room temperature and monitored by a membrane flotation assay the majority of 3a containing membrane structures was found at the bottom of the gradient but a small population of 3a protein was still detectable in the interface of 10 to 50 sucrose Fig 2C These results indicated that at least some of the 3a protein in the membrane structures was resistant to detergent treatment strongly suggesting that a subpopulation of the released 3a protein is associated with detergent resistant membranes 5 7 37 Sucrose density equilibrium ultracentrifugation of the 3a containing membranous structures showed that 3a protein dis played a relatively broad distribution in the gradient ranging from 1 21 to 1 11 g ml Fig 3 fractions 4 to 10 and the peak signal of 3a protein was observed at about 1 14 to 1 16 g ml Fig 3 fractions 7 and 8 which was lighter than the buoyant densities 1 160 to 1 185 g ml of SCoV 17 Release of 3a protein from SCoV infected cells We next tested whether 3a containing membranous structures were produced in SCoV infected cells We previously had analyzed a highly purified SCoV sample and did not observe the pres ence of 3a containing membranous structures with a lighter buoyant density than SCoV 17 In that experiment SCoV was purified by two successive discontinuous sucrose gradient centrifugations for 3 and 18 h and a continuous overnight sucrose gradient centrifugation Although this purification pro cedure is suitable for SCoV purification it might have ex cluded putative 3a containing membranous structures during FIG 1 Release of SCoV 3a protein from 3a protein expressing cells 293T cells were independently transfected with pCAGGS mock pCAGGS 3a 3a or pCAGGS 7a 7a After 48 h transfec tion supernatants from transfected cells were clarified applied onto a 20 sucrose cushion and centrifuged at 26 000 rpm for3hat4 C The pellets were suspended in 1H11003 SDS PAGE loading buffer Medium Cell lysates were prepared with 1H11003 SDS PAGE loading buffer Cell Samples were subjected to Western blotting analysis with anti 3a an tibody A and anti 7a antibody B The membranes were reprobed with anti actin antibody anti actin 3a 1 indicates 3a protein with a molecular mass of H1101137 kDa FIG 2 Flotation assay of 3a containing membranous structures Purified 3a containing membrane structures from the media of 3a expressing 293T cells were suspended in NTE buffer A in NTE buffer containing 1 Triton X 100 and incubated at 4 C for 30 min B or in NTE buffer containing 1 Triton X 100 at room tempera ture and incubated for 30 min C After sucrose was added to adjust the final concentration to 60 the samples were transferred to the bottom of Beckman SW41 centrifuge tubes and overlaid with 50 sucrose and 10 sucrose The step gradient was centrifuged to equi librium at 38 000 rpm for 18 h by using a Beckman SW41 rotor Fractions were collected from top to bottom Equal amounts of each fraction were diluted in NTE buffer separated by SDS PAGE and analyzed by Western blotting with anti 3a antibody 3a 1 and 3a 2 indicate the slow migrating 37 kDa and fast migrating 31 kDa forms of the 3a protein respectively 212 HUANG ET AL J VIROL on March 9 2015 by University of Pittsburgh HSLS http jvi asm org Downloaded from the purification steps In the present study supernatants from SCoV infected Caco2 cells were clarified by low speed centrif ugation and subsequent filtration The released SCoV and putative membrane structures containing 3a protein were pel leted by ultracentrifugation through a 20 sucrose cushion and then applied onto a 20 to 60 continuous sucrose g